Manufacture of surfactant-containing compositions with enhanced stability

ABSTRACT

Improved methods for the manufacture of pharmaceutical compositions comprising at least one surfactant, involving prefiltration of the surfactant prior to formulation into final products.

RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

The present patent application is a continuation of U.S. applicationSer. No. 16/299,372 filed Mar. 12, 2019, now allowed, which is acontinuation of U.S. application Ser. No. 15/531,487 filed May 30, 2017,now U.S. Pat. No. 10,238,739, which application is filed pursuant to 35U.S.C. § 371 as a U.S. National Phase Application of InternationalPatent Application No. PCT/EP2015/078270 filed on Dec. 2, 2015, claimingthe benefit of priority to European Patent Application No. 14195981.7filed on Dec. 2, 2014. The International Application was published as WO2016/087479 on Jun. 9, 2016. The contents of each of the aforementionedpatent applications are herein incorporated by reference in theirentirety.

FIELD OF THE INVENTION

This invention is in the field of manufacturing compositions thatinclude a surfactant, such as pharmaceutical compositions, and, inparticular, achieving the reduction of impurities in such compositions.

BACKGROUND

Certain components of pharmaceutical, nutraceutical and cosmeticcompositions are known to undergo unwanted degradation and/or chemicalalterations, affecting the quality or shelf-life of such products. Forinstance, surfactants can degrade to generate unwanted components, e.g.,polysorbates can yield unwanted carbonyl degradation products.

Surfactants are used in emulsions. The vaccine adjuvant known as “MF59”[References 1-3] is a submicron oil-in-water emulsion of squalene,polysorbate 80 (also known as TWEEN® 80), and sorbitan trioleate (alsoknown as SPAN® 85). It may also include citrate ions, e.g., 10 mM sodiumcitrate buffer. The composition of the emulsion by volume can be about5% squalene, about 0.5% TWEEN® 80 and about 0.5% SPAN® 85. The adjuvantand its production are described in more detail in Chapter 10 ofReference 4, chapter 12 of Reference 5 and chapter 19 of Reference 6.Polysorbate 80 and squalene are also present in the emulsion adjuvantknown as “AS03” [Reference 16].

Polysorbates can undergo autooxidation, generating peroxides, aldehydes,ketones and acids. Other components in a composition, such as proteins,can be affected by some of these degradation products. For instance,conformation of proteins can change, which can affect their efficacy[References 7, 8].

In addition, squalene has been shown to undergo oxidative degradationresulting in the generation of carbonyl by-products. Photo-oxidativecleavage of squalene has been shown to produce formaldehyde,malonaldehyde, acetaldehyde and acetone [References 9], althoughdegradation can be reduced by storage in an air-tight container, storageunder nitrogen, protection from light, etc.

SUMMARY OF THE INVENTION

The present invention provides methods for improving the quality and/orshelf life of compositions that contain at least one surfactant, such asa non-ionic surfactant. The invention encompasses the recognition thatcertain pre-processing of a surfactant component for the manufacture ofa product into which the surfactant becomes incorporated cansignificantly improve the quality of the resulting product. It has beenfound that such pre-processing can in some cases enhance the stabilityof the product, prolong its shelf life, and/or maintain its overallquality. Such effects may be achieved by, for example, prefiltering thesurfactant component (e.g., liquid surfactants, solutions containing asurfactant, etc.) prior to formulating into a product. The inventionthus offers a novel solution for improving the manufacturing processand/or the quality of surfactant-containing products by controlling thequality of an ingredient (e.g., starting material and/or intermediate)comprising a surfactant.

A number of products, in particular, pharmaceutical formulations (seebelow), food and nutraceutical formulations (dietary supplements, foodingredients, etc.), as well as cosmetic formulations (e.g., lotions,creams, gels, foams, etc.), are formulated with at least one surfactant.The present invention therefore may be useful for improving suchproducts.

Pre-processing (such as prefiltration) of a surfactant component isaimed at enhancing the quality of or increasing purity of the componentused as a starting material or an intermediate ingredient, by removingimpurities and/or aggregates that may be present in the raw orunprocessed surfactant. Furthermore, removal of any aggregates is alsobeneficial if the surfactant will later be used in an apparatus whichcan become clogged, such as in a microfluidisation chamber.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 depicts the acetaldehyde content measured in ppm in a number ofbatches of an exemplary surfactant-containing composition. Data pointsin the middle section of the diagram correspond to the batches thatincluded a surfactant which had been pre-filtered, while data points inthe flanking sections correspond to the batches that included asurfactant which had not been pre-filtered. All other productionparameters remained unchanged.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention provides methods for improving the manufacturingprocess and/or the quality of a surfactant-containing composition. Theinvention described herein is therefore suitable for any products thatcomprise at least one surfactant, including, for example, pharmaceuticalcompositions, nutraceutical compositions and cosmetic compositions. Thepresent invention offers a novel solution to improve the manufactureprocess and/or the quality of such compositions by improving the qualityof a surfactant component itself by pretreating the surfactant asdescribed herein. The improved methods are particularly suitable whenthe enhanced stability of the product is desirable.

Thus, the compositions and methods described herein are particularlyuseful for the manufacture of emulsion-based products. Surfactants arewidely used as emulsifiers for scientific and commercial purposes. Anemulsifier may be added to facilitate formation and stability of anemulsion, such as oil-in-water emulsions and water-in-oil emulsions.Emulsion-based products may be prone to destabilization by virtue ofhaving two or more liquid components that are naturally immiscibledispersed therein, which may be thermodynamically unfavorable. Forcertain emulsion-based products that require a high degree ofuniformity, purity, and/or stability, it is especially beneficial toensure that the product does not undergo unwanted degradation oralteration of chemical compositions over time. Accordingly, someembodiments of the invention are drawn to improved methods for theproduction of oil in water emulsions, in particular methods which leadto a reduction in undesirable carbonyl impurities in the finalemulsions.

Surprisingly, the inventor has found that levels of carbonyl impuritiesin oil-in-water emulsions can be reduced by size filtration of asurfactant (e.g., polysorbate 80), and then using this filtered materialto prepare the emulsion. Without wishing to be bound by theory, theinventor believes that carbonyl degradation products in a surfactantmaterial reside preferentially within large aggregates, and that thefiltration removes such aggregates. The filtration can be achieved witha filter having a pore size larger than used in filter sterilization,and it is advantageously performed on non-aqueous surfactant (such asnon-aqueous polysorbate 80) before being dissolved in water, although itis also possible to carry out prefiltration of an intermediate materialthat comprises the surfactant and at least part of an aqueous component.Reducing the levels of carbonyl impurities is particularly important inemulsions which contain other components which can also provide carbonyldegradation products, e.g., squalene. Thus the invention is particularlyuseful for manufacturing emulsion vaccine adjuvants such as “MF59” or“AS03.”

Thus the invention provides bulk preparations of a pre-processedsurfactant containing (i) a carbonyl level that is lower than that ofthe surfactant not subjected to prefiltration; and/or (ii) feweraggregates than the surfactant not subjected to prefiltration. Thephrase “bulk preparation” refers to a commercial-scale preparation ofsuch surfactants. Typically, such a preparation has a volume of at least50 liters, e.g., at least 75 liters, at least 100 liters, at least 125liters, at least 150 liters, at least 175 liters, at least 200 liters,at least 250 liters, or at least 300 liters. Such pre-processedsurfactants can then be used in the manufacture of any suitableproducts.

The invention also provides a process for preparing a surfactantcomprising a step of filtering non-aqueous surfactant through a filterhaving a pore size between 5-50 μm. The filtered surfactant can then beused for downstream purposes, such as those disclosed herein. Theprocess is particularly useful for preparing bulk preparations ofsurfactant. The process is particularly suitable for filteringnon-aqueous polysorbate 80.

In another aspect, the invention provides processes for preparing asurfactant-containing formulation using a pre-processed surfactant(e.g., prefiltered surfactant). “Pre-processed” surfactants may beprovided by a prefiltration step as described herein. For example,prefiltration of a surfactant in accordance with the present inventionmay be carried out with the use of a filter having a pore size between5-50 μm. The prefiltered surfactant may contain a carbonyl level that islower than that of the surfactant not subjected to prefiltration. Theprefiltered surfactant may also contain fewer aggregates than thesurfactant not subjected to prefiltration.

In some embodiments, the invention provides a process for preparing anemulsion (such as an oil-in-water emulsion) using a surfactant that hasbeen pre-processed as described herein, e.g., to achieve reduced levelsof carbonyl impurities and/or fewer aggregates.

In exemplary embodiments, such process may involve the following steps:(a) providing a surfactant, such as polysorbate 80, which has beenprefiltered through a filter having a pore size between 5-50 μm, toprovide purified (e.g., prefiltered) surfactant (e.g., polysorbate 80);and (b) combining the purified surfactant (e.g., polysorbate 80) with anoil component to provide an emulsion. In addition to the purifiedsurfactant and the oil component, the emulsion will include an aqueouscomponent (and, as described in more detail below, possibly furthercomponents as well). Aqueous material may be added as a third componentin step (b), or may be combined with the surfactant and/or the oil priorto step (b), or may even be present in the surfactant filtered in step(a). Thus the surfactant (e.g., polysorbate 80) which is obtained instep (a) can be an aqueous solution of the surfactant (e.g., polysorbate80), but may also be non-aqueous surfactant i.e., surfactant which hasnot been diluted with water (or any other aqueous material) prior tobeing filtered. Thus the surfactant (e.g., polysorbate 80) may befiltered in the “raw” or “bulk” condition in which it arrives from amanufacturer. Typically, step (a) of the process comprises a step offiltering the surfactant.

Similarly, the invention includes a process for preparing anoil-in-water emulsion comprising a step of combining a polysorbate 80with an oil component, wherein the polysorbate 80 has previously beenfiltered through a filter having a pore size between 5-50 μm. Thepolysorbate 80 may have been treated between being filtered and beingcombined with the oil component, e.g., it may have been diluted toprovide an aqueous surfactant. It is understood that the prefiltrationstep and the subsequent formulation step(s) may be carried out at thesame site or in separate sites, and by the same person or by differentpeople.

The invention also provides a process for preparing an oil-in-wateremulsion comprising steps of: (a) filtering non-aqueous polysorbate 80,to provide purified polysorbate 80; and (b) combining the purifiedpolysorbate 80 with an oil component to provide the oil in wateremulsion. The filter used in step (a) can have a pore size between 5-50μm, which is much larger than used for filter sterilization (e.g., 0.22μm). Between steps (a) and (b) the purified polysorbate 80 can becombined with aqueous material (e.g., water or buffer) to give aqueouspurified polysorbate 80 which is then combined with an oil component instep (b).

Similarly, the invention provides a process for preparing anoil-in-water emulsion comprising a step of combining a polysorbate 80with an oil component, wherein the polysorbate 80 has previously beenfiltered in non-aqueous form.

In some embodiments, the invention provides a process for preparing anoil-in-water emulsion comprising steps of: (a) filtering non-aqueouspolysorbate 80 through a filter having a pore size between 5-50 μm, toprovide purified polysorbate 80; and (b) combining the purifiedpolysorbate 80 with an oil component to provide the oil in wateremulsion.

Similarly, the invention provides a process for preparing anoil-in-water emulsion comprising a step of combining a polysorbate 80with an oil component, wherein the polysorbate 80 has previously beenfiltered in non-aqueous form with a filter having a pore size between5-50 μm.

The invention also provides a process for preparing an oil-in-wateremulsion comprising steps of: (a) filtering non-aqueous polysorbate 80through a filter having a pore size between 5-50 μm, to provide purifiednon-aqueous polysorbate 80; (b) combining the purified non-aqueouspolysorbate 80 with an aqueous material, to provide an aqueouscomponent; and (c) combining the aqueous component with an oil componentto provide the oil in water emulsion.

Similarly, the invention provides a process for preparing anoil-in-water emulsion comprising a step of combining an aqueouspolysorbate 80 solution with an oil component, wherein the solution wasformed by mixing an aqueous carrier with polysorbate 80 which hadpreviously been filtered in non-aqueous form with a filter having a poresize between 5-50 μm.

The invention also provides a process for preparing a vaccine,comprising a step of combining an emulsion of the invention with anantigen. Similarly, the invention provides a process for preparing avaccine, comprising steps of: (i) preparing an emulsion as describedherein; and (ii) combining this emulsion with an antigen. In general,however, the invention has broader applicability than just vaccines.

The invention also provides a process for preparing a vaccine,comprising steps of: (i) preparing an emulsion as described above; and(ii) packaging the emulsion into a kit as a kit component together withan antigen component. The antigen and emulsion kit components can thenbe combined at a later time (e.g., at the point of use) foradministration to a patient.

Carbonyl Components

Carbonyl components (e.g. formaldehyde, acetaldehyde and acetone) canarise as degradation products or residual impurities in variousmaterials, such as polysorbate 80 and/or squalene. Their avoidance canbe particularly important in pharmaceutical products, such as inemulsions where polysorbate 80 and/or squalene are present. Thesecarbonyl components can react with other components in a composition(such as a protein) and thus their presence is undesirable. Acetaldehydeis of particular concern because, more than formaldehyde and acetone,its levels have been seen to rise during storage of oil-in-wateremulsions which contain polysorbate 80 and squalene, due to degradationof these two compounds. If levels of acetaldehyde are already high inthe starting materials then this degradation can mean that final levelsat the time of use can be undesirably high.

Filtering polysorbate 80 prior to forming the oil-in-water emulsion hasa beneficial effect. Thus the processes of the invention facilitate theproduction of oil-in-water emulsions which comprise less than 0.85 ppmacetaldehyde, e.g., less than 0.80 ppm, less than 0.75 ppm, less than0.70 ppm, less than 0.65 ppm, less than 0.60 ppm, less than 0.55 ppm,less than 0.50 ppm, less than 0.45 ppm, less than 0.40 ppm, or evenlower. The level in ppm is based on weight, such that 0.85 ppm means0.85 μg acetaldehyde per gram of emulsion. By achieving this low levelof acetaldehyde when the emulsion is first formed then its shelf lifecan be increased from about 3 years to about 5 years, under otherwiseidentical storage conditions.

The invention thus provides an oil-in-water emulsion comprisingpolysorbate 80, wherein the emulsion contains less than 0.85 ppmacetaldehyde, as discussed above. Methods for providing emulsions havingsuch low levels of acetaldehyde have not previously been disclosed. Theemulsion ideally comprises squalene, and further details on the emulsionare disclosed herein.

When stored, acetaldehyde levels in this emulsion can rise over time,but the low starting level and the polysorbate 80 filtration can ensurea long shelf-life with no more than 2.6 ppm acetaldehyde after 3 yearsstorage at 4° C. (and, advantageously, even after 5 years). Thus, in asixth aspect, the invention provides an oil-in-water emulsion comprisingpolysorbate 80, wherein the emulsion contains less than 2.6 ppmacetaldehyde after storage at 4° C. for at least 3 years, e.g., for 5years.

Surfactants

Processes of the invention utilize at least one surfactant, i.e., one ormore surfactants. Suitable surfactants include non-ionic surfactants. Insome embodiments, the surfactant is a polymeric surfactant (i.e., itincludes a polymeric group such as a polyoxyethylene orpolyoxypropylene). In some embodiments, surfactants are non-ionicpolymeric surfactants. In some embodiments, surfactants are non-ionic,polymeric ether surfactants. A preferred class of surfactants for usewith the invention include at least one polyoxyethylene group, such asthe poloxamers and polysorbates e.g. polysorbate 80 (commonly known bythe trade name TWEEN™ 80).

Non-limiting examples of surfactants include TWEEN® 20 (polysorbate 20),TWEEN® 40 (polysorbate 40), TWEEN® 60 (polysorbate 60), TWEEN® 80(polysorbate 80), TRITON™ X-100, IGEPAL CA-630, Nonidet P-40, PLURONIC®F-68, PLURONIC® F-88, and PLURONIC® F-127 (poloxamers), and Brij 35(polyoxyethylene alkyl ether). The most preferred surfactant for usewith the invention is polysorbate 80, which is a polyoxyethylenesorbitan ester surfactant also known as polyoxyethylene (20) sorbitanmonooleate. It is available from a wide range of commercialmanufacturers, including at pharmaceutical grade (e.g., the ultra-pure“Polysorbate (HX2)” product from NOF Corporation), and it is present inthe “MF59” and “AS03” oil in-water emulsion vaccine adjuvants. It isgenerally supplied as a pure clear yellow/brown liquid.

Polysorbate 80 contains ether linkages and unsaturated alkyl chains thathave been shown to auto-oxidize in aqueous solution, leading to reactivealdehydes including acetaldehyde which can cause undesirableside-reactions (e.g., by reacting with primary amino groups onproteins).

Thus the invention aims to reduce levels of acetaldehyde in polysorbate80, and in emulsions which contain polysorbate 80, by utilising thefiltration step disclosed herein. The filtered material is then used forpreparing the oil in water emulsion, thereby providing final emulsionshaving reduced levels of acetaldehyde impurities.

During manufacture of oil in water emulsions with polysorbate 80 it isusual to disperse it in an aqueous carrier, and then to use this aqueousmaterial to form the emulsion. Although the filtration step of theinvention can be performed after polysorbate 80 has been mixed (diluted)with an aqueous carrier (e.g., w.f.i. or buffer), which may be used toreduce viscosity, it is preferred to filter the polysorbate 80 beforesuch mixing occurs (i.e. it is performed on non-aqueous polysorbate 80);it may even be performed on anhydrous material (i.e., havingsubstantially no water content), although the product specification forpolysorbate 80 often permits up to 3 wt % water (which material isnon-aqueous as it has no added water, but is not anhydrous). Thefiltered non-aqueous material can then be diluted with an aqueouscarrier and used in the manner already known in the art (see below). Itis also possible to mix non-aqueous polysorbate 80 with an aqueouscarrier, then filter it, and then add further aqueous carrier to providea final aqueous material ready for emulsification. Usually the materialto be filtered is substantially free from any components other than thepolysorbate 80 and any optional aqueous carrier, in which case thepolysorbate 80 is not mixed with any component (except, optionally, theaqueous carrier) before being filtered.

Size Filtration of Surfactants

The invention utilizes a pre-filtration step for a surfactant. In someembodiments, such surfactant is a component of a product to beformulated. As discussed above, this pre-filtration step has been foundto have a beneficial effect. By having a reduced initial acetaldehydeconcentration the shelf-life of the adjuvant is thereby improved. Thus,filtration of a surfactant in accordance with the invention provides apurified surfactant.

The invention thus includes a prefiltration step for non-ionicsurfactants, such as polysorbate 80. Filtration of the polysorbate 80provides purified polysorbate 80 for use with the invention. Thepurified non-ionic surfactants, such as purified polysorbate 80, containless than 0.85 ppm acetaldehyde. In some embodiments, a non-ionicsurfactant prefiltered in accordance with the present invention containsno more than 0.85 ppm acetaldehyde, no more than 0.80 ppm acetaldehyde,no more than 0.75 ppm acetaldehyde, no more than 0.70 ppm acetaldehyde,no more than 0.65 ppm acetaldehyde, no more than 0.60 ppm acetaldehyde,no more than 0.55 ppm acetaldehyde, no more than 0.50 ppm acetaldehyde,no more than 0.45 ppm acetaldehyde, no more than 0.40 ppm acetaldehyde,no more than 0.35 ppm acetaldehyde, no more than 0.30 ppm acetaldehyde,no more than 0.25 ppm acetaldehyde, no more than 0.20 ppm acetaldehyde,or less.

As mentioned above, a surfactant to be filtered, e.g., polysorbate 80,can be mixed with water, to give aqueous surfactant, e.g., aqueouspolysorbate 80, prior to filtration, but in some embodiments it ispreferred to filter non-aqueous surfactant, e.g., non-aqueouspolysorbate 80.

Filtration of surfactant, e.g., non-ionic surfactants such aspolysorbate 80, utilizes a filter having a pore size which is between 5μm and 50 μm, e.g., between 10-40 μm, between 10-30 μm, between 15-25μm, between 18-22 μm, or about 20 μm. The pore size of a filter isassessed as the diameter of the largest glass particle which will passthrough the filter under normal operating conditions.

In some embodiments, capsule filters are useful. Capsules includeinternal filtration membranes which provide a large surface area withina small volume to permit high throughput and flow rates.

Suitable filters include those formed from polypropylene fleece,polyethersulfone fleece, cellulose acetate, or glass fibre. Particularlyuseful filters include polypropylene fleece filters, e.g., a SARTOPURE™PP 2 MIDICAP™ filter (which is available with pore size of 5 μm, 8 μm,20 μm and 50 μm). A 20 μm polypropylene fleece filter may beparticularly useful, such as the appropriate SARTOPURE™ PP 2 MIDICAP™filter. This filter uses a 20 μm pleated polypropylene depth filterfleece material. Its 20 μm pore size is referred to by the manufactureras its ‘retention rate’, and this filter's operating parameters permit amaximum allowable differential pressure of 5 bar at 20° C., with amaximum allowable back pressure of 2 bar.

It is known that filters often do not operate efficiently until acertain amount of sample has been processed. Monitoring of filtrate canbe used to determine when the filter has settled down and is operatingas desired. A SARTOPURE™ PP 2 MIDICAP™ 20 μm filter typically needs aflow of approximately 200 ml polysorbate 80 before it operates well, andso the first 200 ml or so of filtrate are generally discarded.

Typically, prefiltration of surfactant(s) in accordance with the presentinvention is carried out at room temperature (e.g., ambient condition),which is in the context of the invention defined as between about 14-25°C., more typically between about 18-22° C. In some embodiments, suchprefiltration may be carried out at an elevated temperature, such asbetween about 26-45° C. A peristaltic pump may be used.

The filtered (purified) surfactant, e.g., filtered (purified)polysorbate 80, may be combined with an aqueous carrier to form anaqueous component which can then be used for subsequent step(s) ofmanufacture or formulation. For example, in cases of emulsionproduction, the filtered (purified) surfactant such as polysorbate 80may be used for emulsification of an oil component and an aqueouscomponent, so as to form an emulsion, e.g., oil-in-water emulsions andwater-in-oil emulsions. Suitable aqueous carriers may be plain water(e.g., w.f.i.) or can include further components, e.g., solutes. Forinstance, it may include salts to form a buffer, e.g., citrate orphosphate salts, such as sodium salts. Typical buffers include: aphosphate buffer; a Tris buffer; a borate buffer; a succinate buffer; ahistidine buffer; or a citrate buffer. Buffers will typically beincluded in the 5-50 mM range, but the skilled artisan may choose to useany suitable strength of buffers.

When the invention includes a step of post-filtration pre-emulsificationmixing with an aqueous carrier, this mixing ideally uses an excessvolume of aqueous carrier, e.g., at a volume ratio between 1:40 and 2:5(e.g., from 1:20 to 1:5, 1:15 to 1:7, or about 1:10).

A surfactant will generally be used within 2 years of being filtered(i.e., prefiltered). Thus, for instance, if the surfactant is used toprepare an emulsion (as disclosed herein) then this can occur within 2years of the filtration having occurred. In some embodiments it can beused after shorter durations, e.g., within 18 months, within 12 months,within 9 months, within 6 months, within 3 months, within 1 month,within 2 weeks, within 10 days, within 1 week, within 6 days, within 5days, within 4 days, within 72 hours, within 60 hours, within 48 hours,within 24 hours or less. In some embodiments, a prefiltered surfactantthat was stored longer than a certain duration may be re-filtered (i.e.,repeated prefiltration) prior to being formulated into products ofinterest. For example, a prefiltered surfactant that has been storedlonger than 2 years, 18 months, 12 months, 6 months, 3 months, 2 months,1 month, 3 weeks, 2 weeks, 10 days, 1 week, 6 days, 5 days, 4 days, 72hours, 60 hours, 48 hours, or 24 hours, may be filtered again prior tobeing formulated into a composition, such as emulsions.

According to the invention, compositions formulated with a surfactantprocessed as described herein exhibit enhanced stability. In someembodiments, the shelf-life of such compositions may be increased by atleast 20%, e.g., at least 20%, at least 30%, at least 40%, at least 50%,at least 60%, at least 70%, at least 80%, at least 90%, at least 100%,or greater, as compared to negative control (i.e., equivalentcounterpart that contains the same components but in which at least onesurfactant is not pretreated in accordance with the present inventionand is stored under the same condition).

Emulsions

Processes of the invention are also useful for the manufacture ofemulsions, such as oil in water emulsions, water-in-oil emulsions, orwater-in-oil-in-water emulsions. Thus, the invention provides methodsfor preparing an emulsion comprising at least one filtered (purified)surfactant (i.e., prefiltered prior to formulating into an emulsion).These emulsions, or products formulated with them, are particularlysuitable for parenteral administration for pharmaceutical purposes e.g.vaccine adjuvants. These emulsions include at least three coreingredients: an oil component; an aqueous component; and a surfactantcomponent.

In general, oil-in-water emulsions of the invention are formed bycombining at least one oil (i.e., an oil component), an aqueouscomponent such as water, and at least one surfactant which has beensubjected to the methods described herein. Although spontaneousemulsification is possible, mixing will normally involve mechanical aid,such as the use of a homogenizer and/or a microfluidizer. A usefulemulsification process mixes components first by using a homogenizer andthen by using a microfluidizer. Where a component includes more than onecompound (e.g., two different oils in the oil component, two differentsurfactants in the surfactant component, etc.), these may be combined invarious orders prior to emulsification, e.g., a first surfactant and anoil may be pre-mixed, to be combined with a mixture of a secondsurfactant and an aqueous component.

Details of suitable oil component are discussed further below.

In some embodiments, emulsions of the invention comprise a non-ionicsurfactant. In some embodiments, emulsions of the invention areoil-in-water emulsions which comprise a non-ionic surfactant. The terms“non-ionic” and “nonionic” are used interchangeably herein. In someembodiments, such oil-in-water emulsions comprise more than onesurfactant, at least one of which is optionally a non-ionic surfactantwhich has been processed in accordance with the invention. In someembodiments, the surfactant component of an emulsion comprises apolysorbate which has been processed in accordance with the invention.In some embodiments, the surfactant component of an emulsion comprisespolysorbate 80 which has been processed as discussed herein. Polysorbate80 can be the sole surfactant in an emulsion of the invention, or it ispossible to use multiple surfactants, i.e., comprising filteredpolysorbate 80 and at least one further surfactant. For instance, “AS03”has polysorbate 80 as its sole surfactant component, whereas “MF59” usesa mixture of polysorbate 80 and sorbitan trioleate (“SPAN® 85”).

Surfactants can be classified by their “HLB” (hydrophile/lipophilebalance), where an HLB in the range 1-10 generally means that thesurfactant is more soluble in oil than in water, and an HLB in the range10-20 are more soluble in water than in oil. Polysorbate 80 has a HLB of15.0. If multiple surfactants are used then it will be usual to includea surfactant with an HLB value in the range of 1-10 (e.g., SPAN® 85,with an HLB of 1.8). Surfactants which can be used in addition topolysorbate 80 include, but are not limited to: the polyoxyethylenesorbitan esters surfactants (commonly referred to as the Tweens), suchas polysorbate 20; copolymers of ethylene oxide (EO), propylene oxide(PO), and/or butylene oxide (BO), sold under the DOWFAX™ tradename, suchas linear EO/PO block copolymers; octoxynols, which can vary in thenumber of repeating ethoxy (oxy-1,2-ethanediyl) groups, withoctoxynol-(TRITON™ X-100, or t-octylphenoxypolyethoxyethanol) being ofparticular interest; (octylphenoxy)polyethoxyethanol (IGEPALCA-630/NP-40); phospholipids such as phosphatidylcholine (lecithin);polyoxyethylene fatty ethers derived from lauryl, cetyl, stearyl andoleyl alcohols (known as Brij surfactants), such as triethyleneglycolmonolauryl ether (Brij 30); polyoxyethylene-9-lauryl ether; and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (SPAN®85) and sorbitan monolaurate. A mixture of polysorbate 80 and sorbitantrioleate is particularly useful with the invention, e.g., at a 1:1volume ratio.

Polysorbate 80 is biodegradable (metabolisable) and biocompatible; iffurther surfactant(s) is/are included then this/these may also bebiodegradable and biocompatible.

The aqueous component comprises an aqueous solution which may be plainwater (e.g., w.f.i.) or can include further components, e.g., solute(s).For instance, it may include salts to form a suitable buffer, e.g.,citrate or phosphate salts, such as sodium salts. Typical buffersinclude, but are not limited to: a phosphate buffer, a Tris buffer, aborate buffer, a succinate buffer, a histidine buffer, and a citratebuffer. Buffers will typically be included in the 5-50 mM range,depending on the particular buffer being used.

In some embodiments, the total amount of oil(s) (% by volume) in thefinal emulsion is between about 1-25%, e.g., about 1%, about 2%, about3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%,about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about23%, about 24%, about 25%. In some embodiments, the total amount ofoil(s) (% by volume) in the final emulsion is between about 2-20%,between about 4-15%, between about 8-12%, for example, about 10%. Whenan emulsion comprises squalene as an oil component, a squalene content(% by volume) of between about 5% and about 10% is particularly useful,e.g., about 5%. In some embodiments, an emulsion comprises a squalenecontent (w/v) of between about 30-50 mg/ml, e.g., between about 35-45mg/ml, about 36-43 mg/ml, about 38-40 mg/ml, etc.

In some embodiments, amounts of surfactants (% by weight) in the finalemulsion are: polyoxyethylene sorbitan esters (such as TWEEN® 80) 0.02to 2%, in particular about 0.5% or about 1% or about 2%; sorbitan esters(such as SPAN® 85) 0.02 to 2%, in particular about 0.5% or about 1%;octyl- or nonylphenoxy polyoxyethanols (such as TRITON™ X-100) 0.001 to0.1%, in particular 0.005 to 0.02%; polyoxyethylene ethers (such aslaureth 9) 0.1 to 20%, preferably 0.1 to 10% and in particular 0.1 to 1%or about 0.5%. A polysorbate 80 content (w/v) of between 4-6 mg/ml isuseful, e.g., between 4.1-5.3 mg/ml, but higher levels are also useful,e.g., from 19-20 mg/ml. A sorbitan trioleate content (w/v) of between4-6 mg/ml is useful, e.g., between 4.1-5.3 mg/ml.

The emulsion comprises polysorbate 80 and squalene and contains lessthan 0.85 ppm acetaldehyde.

Emulsions of the invention may be prepared in accordance with thetechniques variously disclosed in References 10, 11 and 12 (the completecontents of each of which are incorporated herein by reference). Thusemulsification may involve formation of a first emulsion byhomogenization, followed by microfluidization to provide a secondemulsion. Components may be recirculated through the homonogenizerand/or the microfluidiser using “type II” circulation [Reference 11], ora combination of “type I” and “type II” circulation. An inert atmospherecan be used during mixing. Temperature can be controlled around 40° C.,for example, between about 35° C. and 45° C., e.g., about 35° C., about36° C., about 37° C., about 38° C., about 38° C., about 40° C., about41° C., about 42° C., about 43° C., about 44° C., and about 45° C. Theemulsions can be filtered using a sterile asymmetric hydrophilic PESmembrane. In some embodiments, the emulsions may be filtered using asterile dual-layer (double-layer) 0.45 μm/0.22 μm asymmetric hydrophilicPES membrane filter.

Emulsions of the invention ideally have an average oil droplet diameterbelow 200 nm. In some embodiments, an average oil droplet of suchemulsions has a diameter of, between about 50 nm and 200 nm, e.g., about50 nm, about 60 nm, about 70 nm, about 800 nm, about 90 nm, about 100nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, and about200 nm. In some embodiments, a diameter of oil droplets that is 155±20nm is typical, and it will generally not be less than 50 nm. The averagediameter (i.e., the number average diameter of the emulsion's oildroplets) may be measured using a dynamic light scattering technique, asdescribed for example in Reference 13. An example of a dynamic lightscattering measurement machine is the Nicomp 380 Submicron Particle SizeAnalyzer (from Particle Sizing Systems).

In some embodiments, surfactants processed by the methods of the presentinvention are useful for producing uniform emulsions, e.g., uniformoil-in-water emulsions and uniform water-in-oil emulsions. Uniformity ofan emulsion refers to having fewer oil droplets that fall outside of apreferred size range within a population of oil droplets. In someembodiments, a uniform oil-in-water emulsion that incorporates at leastone surfactant processed by the methods of the invention contains ≤5%(e.g., ≤4%, ≤3%, ≤2% and ≤1%) of the number of oil droplets that falloutside of a preferred range of diameters. In some embodiments, auniform oil-in-water emulsion that incorporates at least one surfactantprocessed by the methods of the invention is characterized in that thenumber of oil droplets in a population (e.g., preparation) having adiameter of >1.2 μm is 5×10⁸/mL or less, e.g., 5×10⁸/mL or less,5×10⁷/mL or less, 5×10⁶/mL or less, and 5×10⁵/mL or less.

Methods provided herein may be used at large scale, suitable forcommercial-scale production of a composition comprising a surfactantthat has been prefiltered according to the methods embraced in thepresent application. Thus a method may involve preparing a finalemulsion with a volume greater than 1 liter, e.g., >5 liters, >10liters, >20 liters, >50 liters, >100 liters, >250 liters, etc.

The process is particularly useful for preparing any of the followingexemplary oil-in-water emulsions:

An Emulsion Comprising Squalene, Polysorbate 80 (TWEEN® 80), andSorbitan Trioleate (SPAN® 85):

The composition of the emulsion by volume can be about 5% squalene,about 0.5% polysorbate 80 and about 0.5% sorbitan trioleate. In weightterms, these amounts become 4.3% squalene, 0.5% polysorbate 80 and 0.48%sorbitan trioleate. This adjuvant is known as “MF59.” The MF59 emulsionmay advantageously include citrate ions, e.g., 10 mM sodium citratebuffer. In some embodiments, the final concentration of each of suchcomponents present in a product may be halved, e.g., about 2.5%squalene, about 0.25% polysorbate 80 and about 0.25% sorbitan trioleateby volume (about 2.15% squalene, 0.25% polysorbate 80 and 0.24% sorbitantrioleate in weight terms). In some embodiments, the final concentrationof each of such components present in a product may be quartered, e.g.,about 1.25% squalene, about 0.125% polysorbate 80 and about 0.125%sorbitan trioleate by volume (about 1.075% squalene, 0.125% polysorbate80 and 0.12% sorbitan trioleate in weight terms).

As described in Reference 14, MF59 is manufactured on a commercial scaleby dispersing SPAN® 85 in the squalene phase (the oil component) andTWEEN® 80 in the aqueous component, followed by high-speed mixing toform a coarse emulsion (i.e., first emulsion). This coarse emulsion isthen passed through a microfluidizer to produce an emulsion having auniform oil droplet size (i.e., second emulsion). As described inReference 6, the microfluidized emulsion is then filtered through asuitable membrane filter in order to remove any large oil droplets, andthe mean droplet size of the resulting uniform emulsion remainsunchanged for at least 3 years at 4° C. The squalene content of thefinal emulsion can be measured as described in Reference 15.

An Emulsion Comprising Squalene, an α Tocopherol (Ideally DL αTocopherol), and Polysorbate 80:

The emulsion may include phosphate buffered saline. These emulsions mayhave by volume from 2 to 10% squalene, from 2 to 10% tocopherol and from0.3 to 3% polysorbate 80, e.g., 4.3% squalene, 4.7% a tocopherol, 1.9%polysorbate 80. The weight ratio of squalene:tocopherol is preferably <1(e.g., 0.90) as this can provide a more stable emulsion. Squalene andpolysorbate 80 may be present volume ratio of about 5:2 or at a weightratio of about 11:5. Thus the three components (squalene, tocopherol,polysorbate 80) may be present at a weight ratio of 1068:1186:485 oraround 55:61:25. One such emulsion (“AS03” [Reference 16]) has 4.86 mgpolysorbate 80, 10.69 mg squalene and 11.86 mg a tocopherol per dose (ora fraction thereof, but maintaining the mass ratios), e.g., in a 0.5 mlvolume. AS03 can be made by dissolving TWEEN® 80 in PBS to give a 2%solution, then mixing 90 ml of this solution with a mixture of (5 g ofDL α-tocopherol and 5 ml squalene), then microfluidising the mixture.The resulting emulsion may have submicron oil droplets e.g. with anaverage diameter of between 100 and 250 nm, preferably about 180 nm. Theemulsion may also include a 3-de-O-acylated monophosphoryl lipid A (3dMPL). Another useful emulsion of this type may comprise, per human dose,0.5-10 mg squalene, 0.5-11 mg tocopherol, and 0.1-4 mg polysorbate 80[Reference 17], e.g., in the ratios discussed above.

An Emulsion Comprising Squalene, Polysorbate 80, a Triton Detergent(e.g., Triton X 100) and a Tocopherol (e.g., an α-Tocopherol Succinate):

The emulsion may include these three components at a mass ratio of about75:11:10 (e.g., 750 μg/ml polysorbate 80, 110 μg/ml Triton X-100 and 100μg/ml α-tocopherol succinate), and these concentrations should includeany contribution of these components from antigens. The emulsion mayalso include a 3d MPL. The emulsion may also include a saponin, such asQS21. The aqueous component may contain a phosphate buffer.

The compositions of these emulsions, expressed above in percentageterms, may be modified by dilution or concentration (e.g., by aninteger, such as 2 or 3 or by a fraction, such as ⅔ or ¾), in whichtheir ratios stay the same. For instance, a 2-fold concentrated MF59would have about 10% squalene, about 1% polysorbate 80 and about 1%sorbitan trioleate. Concentrated forms can be diluted (e.g., with anantigen solution) to give a desired final concentration of emulsion.

Emulsions of the invention are ideally stored at between 2° C. and 8° C.They should not be frozen. They should ideally be kept out of directlight. In particular, squalene-containing compositions, such asemulsions and vaccines of the invention should be protected to avoidphotochemical breakdown of squalene. If emulsions of the invention arestored then this is preferably in an inert atmosphere, e.g., N2 orargon.

Oil Component

Because emulsions that contain at least one surfactant processed by themethods of the invention are intended to include pharmaceutical use, insuch embodiments, the oil(s) in the emulsion's oil component willtypically be biodegradable (metabolisable) and biocompatible.

As discussed above, the invention is particularly suitable for use inpreparing emulsions which include squalene. Squalene is a naturallyoccurring oil (for example in shark liver) which is a branched,unsaturated terpenoid (C₃₀H₅₀; [(CH₃)₂C[═CHCH₂CH₂C(CH₃)]₂═CHCH₂—]₂;2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene; CAS RN7683-64-9). Squalene is typically purified from animal sources, such asshark, but it is also possible to use squalene from non-animal sources,e.g., genetically-engineered yeasts [Reference 18] and vegetable oilssuch as olive oil [References 19, 20]. Further possible sources includewheat germ oil, palm oil, amaranth seed (e.g., see Reference 21), andrice bran oil. It can be purified by distillation, e.g., as disclosed inReference 22.

The oil component in an emulsion of the invention may include a singleoil, or may comprise a combination of oils, e.g., comprising squaleneand at least one further oil. For instance, “MF59” has squalene as itssole oil component, whereas “AS03” has squalene and α-tocopherol.

Rather than (or on addition to) using squalene an emulsion can compriseoil(s) including those from, for example, an animal (such as fish) or avegetable source. Sources for vegetable oils include nuts, seeds andgrains. Peanut oil, soybean oil, coconut oil, and olive oil, the mostcommonly available, exemplify the nut oils. Jojoba oil can be used e.g.obtained from the jojoba bean. Seed oils include safflower oil,cottonseed oil, sunflower seed oil, sesame seed oil and the like. In thegrain group, corn oil is the most readily available, but the oil ofother cereal grains such as wheat, oats, rye, rice, teff, triticale andthe like may also be used. 6-10 carbon fatty acid esters of glycerol and1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and so may be used. The procedures forseparation, purification, saponification and other means necessary forobtaining pure oils from animal sources are well known in the art.

Most fish contain metabolizable oils which may be readily recovered. Forexample, cod liver oil, shark liver oils, and whale oil such asspermaceti exemplify several of the fish oils which may be used herein.A number of branched chain oils are synthesized biochemically in5-carbon isoprene units and are generally referred to as terpenoids.Squalane, the saturated analog to squalene, can also be used. Fish oils,including squalene and squalane, are readily available from commercialsources or may be obtained by methods known in the art.

Other useful oils are the tocopherols, particularly in combination withsqualene. Where the oil component of an emulsion includes a tocopherol,any of the α, β, γ, δ, ε or ξ tocopherols can be used, but α tocopherolsare preferred. D a tocopherol and DL α tocopherol can both be used. Apreferred α tocopherol is DL α tocopherol. The tocopherol can takeseveral forms, e.g. different salts and/or isomers. Salts includeorganic salts, such as succinate, acetate, nicotinate, etc. If a salt ofthis tocopherol is to be used, the preferred salt is the succinate. Anoil combination comprising squalene and α tocopherol (e.g. DLα-tocopherol) is particularly useful with the invention.

Where the invention utilises shark-derived squalene, any suitable sharkspecies can be used, such as the spiny dogfish (Squalus acanthias).Other suitable species include Centrophorus atromarginatus, Cetorhinusmaximus, Echinorhinus brucus, Scymnodon squamulosus, Centrophorusatromarginatus, Etmopterus frontimaculatus, Deania eglantine andScymnodon foliaceus.

As discussed in Reference 23, when using shark-derived squalene it ispreferred that polychlorinated biphenyls (PCBs) are present at a levelof less than 661 pg PCBs per g squalene (TEQ). This 661 pg/g limit canbe applied to the squalene used to make an emulsion, to the finalemulsion, and to a vaccine made using the emulsion. Usually it appliesto the squalene used to make an emulsion. Full details of how to achievea PCB level within this threshold are found in Reference 23. Levels muchlower than 661 pg/g can be achieved, even as low as ˜2 pg/g.

PCBs are a family of chemical compounds formed by the addition ofchlorine to the 10 positions available for chlorine substitution in abiphenyl ring. Any single chemical compound in the PCB category iscalled a “congener.” Levels of individual congeners are converted to atoxic equivalent (TEQ) which allows the toxicity of a mixture of PCBs tobe represented as a single number. The toxicity of each PCB is expressedas a fraction (the toxic equivalency factor, TEF) of the toxicity of2,3,7,8-TCDD dioxin (which has a reference value of 1). To calculate thetotal TEQ of a mixture, the mass of each PCB is multiplied by its TEFand then the TEQ is the sum of these values.

In some embodiments the squalene has, in addition to a TEQ as above, nodetectable amounts of one or more of: PCB #126; PCB #169; PCB #77; PCB#81; PCB #123; PCB #170; and/or PCB #180. These PCBs are defined byIUPAC nomenclature e.g. PCB #126 is 3,3′,4,4′,5-pentachlorobiphenyl.

As well as having a low PCB content, it is useful to ensure low dioxinlevels. Thus each of the following may have a low dioxin content:squalene used to form emulsions; the final pharmaceutical emulsion; anda vaccine made using the emulsion. A low dioxin content is less than 1ng per gram of squalene, and preferably less than 1 pg/g (TEQ).

Similarly it is useful to ensure low furan levels. Thus each of thefollowing may have a low furan content: squalene used to form emulsionsof the invention; the final pharmaceutical emulsion; and a vaccine madeusing the emulsion of the invention. A low furan content is less than 1ng per gram of squalene, and preferably <1 pg/g (TEQ).

Vaccines

Although it is possible to administer oil in water emulsion adjuvants ontheir own to patients (e.g., to provide an adjuvant effect for anantigen that has been separately administered to the patient), it ismore usual to admix the adjuvant with an antigen prior toadministration, to form an immunogenic composition, e.g., a vaccine.Mixing of emulsion and antigen may take place extemporaneously, at thetime of use, or can take place during vaccine manufacture, prior tofilling. The methods of the invention can be applied in both situations.

Thus a process of the invention may include a further process step ofadmixing the emulsion with an antigen component. As an alternative, itmay include a further step of packaging the adjuvant into a kit as a kitcomponent together with an antigen component.

Overall, therefore, the invention can be used when preparing mixedvaccines or when preparing kits including antigen and adjuvant ready formixing. Where mixing takes place during manufacture then the volumes ofbulk antigen and emulsion that are mixed will typically be greater than1 liter, e.g., >5 liters, >10 liters, >20 liters, >50 liters, >100liters, >250 liters, etc. Where mixing takes place at the point of usethen the volumes that are mixed will typically be smaller than 1milliliter, e.g., <0.6 ml, <0.5 ml, <0.4 ml, <0.3 ml, <0.2 ml, etc. Inboth cases it is usual for substantially equal volumes of emulsion andantigen solution to be mixed i.e. substantially 1:1 (e.g., between 1.1:1and 1:1.1, preferably between 1.05:1 and 1:1.05, and more preferablybetween 1.025:1 and 1:1.025). In some embodiments, however, an excess ofemulsion or an excess of antigen may be used [Reference 24]. Where anexcess volume of one component is used, the excess will generally be atleast 1.5:1, e.g., >2:1, >2.5:1, >3:1, >4:1, >5:1, etc.

Where antigen and adjuvant are presented as separate components within akit, they are physically separate from each other within the kit, andthis separation can be achieved in various ways. For instance, thecomponents may be in separate containers, such as vials. The contents oftwo vials can then be mixed when needed, e.g., by removing the contentsof one vial and adding them to the other vial, or by separately removingthe contents of both vials and mixing them in a third container.

In another arrangement, one of the kit components is in a syringe andthe other is in a container such as a vial. The syringe can be used(e.g., with a needle) to insert its contents into the vial for mixing,and the mixture can then be withdrawn into the syringe. The mixedcontents of the syringe can then be administered to a patient, typicallythrough a new sterile needle. Packing one component in a syringeeliminates the need for using a separate syringe for patientadministration.

In another preferred arrangement, the two kit components are heldtogether but separately in the same syringe, e.g., a dual chambersyringe. When the syringe is actuated (e.g., during administration to apatient) then the contents of the two chambers are mixed. Thisarrangement avoids the need for a separate mixing step at time of use.

The contents of the various kit components will generally all be inliquid form. In some arrangements, a component (typically the antigencomponent rather than the emulsion component) is in dry form (e.g., in alyophilized form), with the other component being in liquid form. Thetwo components can be mixed in order to reactivate the dry component andgive a liquid composition for administration to a patient. A lyophilizedcomponent will typically be located within a vial rather than a syringe.Dried components may include stabilizers such as lactose, sucrose ormannitol, as well as mixtures thereof, e.g., lactose/sucrose mixtures,sucrose/mannitol mixtures, etc. One possible arrangement uses a liquidemulsion component in a pre-filled syringe and a lyophilized antigencomponent in a vial.

If vaccines contain components in addition to emulsion and antigen thenthese further components may be included in one these two kitcomponents, or may be part of a third kit component.

Suitable containers for mixed vaccines of the invention, or forindividual kit components, include vials and disposable syringes. Thesecontainers should be sterile.

Where a composition/component is located in a vial, the vial ispreferably made of a glass or plastic material. The vial is preferablysterilized before the composition is added to it. To avoid problems withlatex sensitive patients, vials are preferably sealed with a latex-freestopper, and the absence of latex in all packaging material ispreferred. In one embodiment, a vial has a butyl rubber stopper. Thevial may include a single dose of vaccine/component, or it may includemore than one dose (a “multidose” vial), e.g., 10 doses. In oneembodiment, a vial includes 10×0.25 ml doses of emulsion. Preferredvials are made of colorless glass.

A vial can have a cap (e.g., a Luer lock) adapted such that a pre filledsyringe can be inserted into the cap, the contents of the syringe can beexpelled into the vial (e.g., to reconstitute lyophilized materialtherein), and the contents of the vial can be removed back into thesyringe. After removal of the syringe from the vial, a needle can thenbe attached and the composition can be administered to a patient. Thecap is preferably located inside a seal or cover, such that the seal orcover has to be removed before the cap can be accessed.

Where a composition/component is packaged into a syringe, the syringewill not normally have a needle attached to it, although a separateneedle may be supplied with the syringe for assembly and use. Safetyneedles are preferred. 1-inch 23-gauge, 1-inch 25-gauge and ⅝-inch25-gauge needles are typical. Syringes may be provided with peel-offlabels on which the lot number, influenza season and expiration date ofthe contents may be printed, to facilitate record keeping. The plungerin the syringe preferably has a stopper to prevent the plunger frombeing accidentally removed during aspiration. The syringes may have alatex rubber cap and/or plunger. Disposable syringes contain a singledose of vaccine. The syringe will generally have a tip cap to seal thetip prior to attachment of a needle, and the tip cap is preferably madeof a butyl rubber. If the syringe and needle are packaged separatelythen the needle is preferably fitted with a butyl rubber shield.

The emulsion may be diluted with a buffer prior to packaging into a vialor a syringe. Typical buffers include: a phosphate buffer; a Trisbuffer; a borate buffer; a succinate buffer; a histidine buffer; or acitrate buffer. Dilution can reduce the concentration of the adjuvant'scomponents while retaining their relative proportions, e.g., to providea “half-strength” adjuvant.

Containers may be marked to show a half dose volume, e.g., to facilitatedelivery to children. For instance, a syringe containing a 0.5 ml dosemay have a mark showing a 0.25 ml volume.

Where a glass container (e.g., a syringe or a vial) is used, then it ispreferred to use a container made from a borosilicate glass rather thanfrom a soda lime glass.

Various antigens can be used with oil in water emulsions, including butnot limited to: viral antigens, such as viral surface proteins;bacterial antigens, such as protein and/or saccharide antigens; fungalantigens; parasite antigens; and tumor antigens. The invention isparticularly useful for vaccines against influenza virus, HIV, hookworm,hepatitis B virus, herpes simplex virus, rabies, respiratory syncytialvirus, cytomegalovirus, Staphylococcus aureus, Chlamydia, SARScoronavirus, varicella zoster virus, Streptococcus pneumoniae, Neisseriameningitidis, Mycobacterium tuberculosis, Bacillus anthracis, EpsteinBarr virus, human papillomavirus, etc. Preferably the antigen is aninfluenza virus antigen. Non-limiting examples of suitable antigens arefurther provided below.

Influenza Virus Antigens: These may take the form of a live virus or aninactivated virus. Where an inactivated virus is used, the vaccine maycomprise whole virion, split virion, or purified surface antigens(including hemagglutinin and, usually, also including neuraminidase).Influenza antigens can also be presented in the form of virosomes. Theantigens may have any hemagglutinin subtype, selected from H1, H2, H3,H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 and/or H16. Vaccinemay include antigen(s) from one or more (e.g., 1, 2, 3, 4 or more)influenza virus strains, including influenza A virus and/or influenza Bvirus, e.g., a monovalent A/H5N1 or A/H1N1 vaccine, or a trivalentA/H1N1+A/H3N2+B vaccine. The influenza virus may be a reassortantstrain, and may have been obtained by reverse genetics techniques [e.g.,References 25-29]. Thus the virus may include one or more RNA segmentsfrom a A/PR/8/34 virus (typically 6 segments from A/PR/8/34, with the HAand N segments being from a vaccine strain, i.e., a 6:2 reassortant).The viruses used as the source of the antigens can be grown either oneggs (e.g., embryonated hen eggs) or on cell culture. Where cell cultureis used, such host cells will typically be an eukaryotic host/cell,e.g., mammalian cells, avian cells, insect cells, plant cells and yeastcells. In some embodiments, suitable host cells are embryonic stemcells. In some embodiments, suitable host cells are of epithelialorigin. In some embodiments, suitable host cells are kidney cells.Suitable mammalian cell lines include, but not limited to: MDCK; CHO;293T; BHK; Vero; MRC 5; PER.C6; WI-38; etc. Suitable avian cell linesinclude, but not limited to: EB66® cells. Preferred mammalian cell linesfor growing influenza viruses include: MDCK cells [References 30-33],derived from Madin Darby canine kidney; Vero cells [References 34-36],derived from African green monkey kidney; or PER.C6 cells [Reference37], derived from human embryonic retinoblasts. Where virus has beengrown on a mammalian cell line then the composition will advantageouslybe free from egg proteins (e.g., ovalbumin and ovomucoid) and fromchicken DNA, thereby reducing allergenicity. Unit doses of vaccine aretypically standardized by reference to hemagglutinin (HA) content,typically measured by SRID. Existing vaccines typically contain about 15μg of HA per strain, although lower doses can be used, particularly whenusing an adjuvant. Fractional doses such as ½ (i.e., 7.5 μg HA perstrain), ¼ and ⅛ have been used [References 38, 39], as have higherdoses (e.g., 3× or 9× doses [References 40, 41]). Thus vaccines mayinclude between 0.1 and 150 μg of HA per influenza strain, preferablybetween 0.1 and 50 μg, e.g., 0.1-20 μg, 0.1 15 μg, 0.1 10 μg, 0.1 7.5μg, 0.5-5 μg, etc. Particular doses include, e.g., about 15, about 10,about 7.5, about 5, about 3.8, about 3.75, about 1.9, about 1.5 μg ofHA, etc. per strain.

Human Immunodeficiency Virus: Suitable antigens include those derivedfrom HIV 1 and HIV 2. The antigen will typically be an envelope antigen.

Hepatitis B Virus Surface Antigens: This antigen is preferably obtainedby recombinant DNA methods, e.g., after expression in a Saccharomycescerevisiae yeast. Unlike native viral HBsAg, the recombinantyeast-expressed antigen is non-glycosylated. It can be in the form ofsubstantially-spherical particles (average diameter of about 20 nm),including a lipid matrix comprising phospholipids. Unlike native HBsAgparticles, the yeast-expressed particles may includephosphatidylinositol. The HBsAg may be from any of subtypes ayw1, ayw2,ayw3, ayw4, ayr, adw2, adw4, adrq− and adrq+.

Hookworm: Suitable hookworm antigens include those particularly as seenin canines (Ancylostoma caninum). Such antigen may be recombinantAc-MTP-1 (astacin-like metalloprotease) and/or an aspartic hemoglobinase(Ac APR 1), which may be expressed in a suitable host expression system,such as baculovirus/insect cell system as a secreted protein [References42, 43].

Herpes Simplex Virus Antigens (HSV): A preferred HSV antigen for usewith the invention is membrane glycoprotein gD. It is preferred to usegD from a HSV 2 strain (“gD2” antigen). The composition can use a formof gD in which the C terminal membrane anchor region has been deleted[Reference 44], e.g., a truncated gD comprising amino acids 1 306 of thenatural protein with the addition of aparagine and glutamine at theC-terminus. This form of the protein includes the signal peptide whichis cleaved to yield a mature 283 amino acid protein. Deletion of theanchor allows the protein to be prepared in soluble form.

Human papillomavirus antigens (HPV): Preferred HPV antigens for use withthe invention are Li capsid proteins, which can assemble to formstructures known as virus-like particles (VLPs). The VLPs can beproduced by recombinant expression of Li in yeast cells (e.g., in S.cerevisiae) or in insect cells (e.g., in Spodoptera cells, such as S.frugiperda, or in Drosophila cells). For yeast cells, plasmid vectorscan carry the Li gene(s); for insect cells, baculovirus vectors cancarry the Li gene(s). More preferably, the composition includes Li VLPsfrom both HPV-16 and HPV-18 strains. This bivalent combination has beenshown to be highly effective [Reference 45]. In addition to HPV 16 andHPV 18 strains, it is also possible to include Li VLPs from HPV 6 andHPV 11 strains. The use of oncogenic HPV strains is also possible. Avaccine may include between 20-60 μg/ml (e.g., about 40 μg/ml) of Li perHPV strain.

Anthrax Antigens: Anthrax is caused by Bacillus anthracis. Suitable B.anthracis antigens include A-components (lethal factor (LF) and edemafactor (EF)), both of which can share a common B-component known asprotective antigen (PA). The antigens may optionally be detoxified.Further details can be found in the literature, e.g., [References46-48].

S. aureus antigens: A variety of S. aureus antigens are known. Suitableantigens include capsular saccharides (e.g., from a type 5 and/or type 8strain) and proteins (e.g., IsdB, Hla, etc.). Capsular saccharideantigens are ideally conjugated to a carrier protein.

S. pneumoniae antigens: A variety of S. pneumoniae antigens are known.Suitable antigens include capsular saccharides (e.g., from one or moreof serotypes 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, and/or 23F) and proteins(e.g., pneumolysin, detoxified pneumolysin, polyhistidine triad proteinD (PhtD), etc.). Capsular saccharide antigens are ideally conjugated toa carrier protein.

A solution of the antigen will normally be mixed with the emulsion,e.g., at a 1:1 volume ratio. This mixing can either be performed by avaccine manufacturer, prior to filling, or can be performed at the pointof use, by a healthcare worker.

Pharmaceutical Compositions

Compositions made using a surfactant processed by the methods of theinvention include pharmaceutical, nutraceutical and cosmeticcompositions. The invention is not limited by a particular type ofproducts, provided that it is desirable to use one or more surfactantsprepared in accordance with the methods provided herein to formulatesuch products. In some embodiments, such products exhibit higher quality(e.g., improved purity, stability, shelf life, or any combinationthereof), as compared to a reference counterpart (control), whichcontains equivalent components or ingredients with the exception of oneor more surfactants not subjected to the methods described herein (e.g.,prefiltration). Thus, in some embodiments, the compositions comprisingone or more surfactants prepared in accordance with the inventioncontain lower levels of carbonyl impurities, as compared to thereference counterpart (control).

In some embodiments, such compositions of the present invention arepharmaceutically acceptable. In relation to vaccines for immunization, acomposition may include one or more additional components, such as anantigen. In some embodiments, such immunization is carried out inconjunction with the use of an adjuvant, e.g., oil-in-water adjuvants.Thus, such vaccines may contain an emulsion-based adjuvant, which mayinclude at least one surfactants processed in accordance with themethods described herein, and an optional antigen.

The composition may include a preservative such as thiomersal or2-phenoxyethanol. It is preferred, however, that such a compositionshould be substantially free from (i.e., less than 5 μg/ml) mercurialmaterial e.g., thiomersal-free [References 49, 50]. Compositions andcomponents containing no mercury are more preferred. For example,preservative-free vaccines are also useful.

The pH of a composition will generally be between 5.0 and 8.1, and moretypically between 6.0 and 8.0, e.g., between 6.5 and 7.5. A process ofthe invention may therefore include a step of adjusting the pH of thecomposition prior to packaging.

The composition is preferably sterile. The composition is preferably nonpyrogenic, e.g., containing <1 EU (endotoxin unit, a standard measure)per dose, and preferably <0.1 EU per dose. The composition is preferablygluten free.

The invention includes kits and compositions prepared using the methodsof the invention. In some embodiments, such compositions areprophylactic compositions, such as vaccines. In any of the embodiments,pharmaceutical compositions comprising a surfactant processed by themethods provided herein may provide improved stability (e.g., longershelf life) as compared to equivalent compositions comprising the samesurfactant but not processed in accordance with the invention.

The composition may include material for a single administrationimmunization, or may include material for multiple administrationsimmunizations (i.e., a “multidose” kit). Where the composition is avaccine for immunization, the vaccine may include material for a singleimmunization or may include material for multiple immunizations (i.e., a“multidose” kit). In some embodiments, the inclusion of a preservativemay be preferred in multidose arrangements.

Compositions containing one or more surfactants processed by the methodsdisclosed herein may be filled in sterile closed systems. Suitablesterile closed systems include, but are not limited to: vials, syringes(such as disposable syringes), glass and plastic containers, includingrigid or flexible containers (e.g., bags). In some embodiments, asterile closed system suitable for use in the present disclosure iscoated for surface-modified, e.g., siliconized.

Methods of Treatment, and Administration of the PharmaceuticalComposition

The compositions prepared according to the methods of the invention aresuitable for administration to subjects, including human patients.Pharmaceutical compositions comprising a surfactant processed by themethods provided herein are suitable for administration to a subject inneed thereof. In some embodiments, such a composition is a therapeuticcomposition that is administered to a subject for the treatment ofdisease or disorder, wherein the composition comprises a suitable activeingredient intended for the treatment. In some embodiments, the immuneresponse is elicited in order to provide therapeutic effects,prophylactic effects, or both. In some embodiments, the immune responseis elicited in order to provide therapeutic effects. In someembodiments, the immune response is elicited in order to provideprophylactic effects, for example, as a vaccine. In some embodiments,such immune response is augmented by the presence of at least oneadjuvant.

In some embodiments, the invention provides a method of raising animmune response in a patient, comprising the step of administering tothe patient a composition of the invention which includes an immunogen,so as to enhance an immune response.

Pharmaceutical compositions prepared according to the invention may beused to treat a subject (e.g., patients) in need thereof, includingchildren and adults, who meet certain criteria, such as clinicalcriteria, genetic criteria, risk factors, age groups, etc. The patientmay be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 yearsold, or at least 55 years old. The patient may be elderly (e.g., >50years old, preferably >65 years), the young (e.g., <5 years old),hospitalized patients, healthcare workers, armed service and militarypersonnel, pregnant women, the chronically ill, immunodeficient orimmunocompromised patients, and people travelling abroad. Wherepharmaceutical compositions are prophylactic vaccines for immunization,such vaccines are suitable not solely for these groups, however, but maybe used more generally in a population, e.g., healthy subjects.

Vaccines of the invention may be administered to patients atsubstantially the same time as (e.g., during the same medicalconsultation or visit to a healthcare professional) other vaccines.

The compositions can be administered in any suitable way, including, butnot limited to: intramuscular injection (e.g., into the arm or leg),subcutaneous injection, intranasal [References 51-53], oral [Reference54], intradermal [References 55, 56], transcutaneous, transdermal[Reference 57], etc.

Pharmaceutical compositions, including vaccines, are typicallyadministered in a dosage volume of about 0.5 ml, although a half dose(e.g., about 0.25 ml) may be administered to children. However,administration volumes are readily adjusted by the person skilled in theart to suit the particular use or purpose.

The invention also provides these kits and compositions for use asmedicaments.

The invention also provides the use of: (i) an aqueous preparation of anantigen or a therapeutic protein or peptide; and (ii) an oil in wateremulsion comprising a surfactant prepared according to the invention, inthe manufacture of a medicament for administering to a patient.

The immune response raised by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.

General

Throughout the specification, including the claims, where the contextpermits, the term “comprising” and variants thereof such as “comprises”are to be interpreted as including the stated element (e.g., integer) orelements (e.g., integers) without necessarily excluding any otherelements (e.g., integers). Thus a composition “comprising” X may consistexclusively of X or may include something additional, e.g., X+Y.

The word “substantially” does not exclude “completely,” e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention. In some embodiments, “a compositionthat is substantially free of a component” may include such acomposition with a residual amount of the component, wherein theresidual amount is (i) below reliable detection levels by suitable orart-accepted measurement means; and/or (ii) the residual amount does notadversely affect an intended purpose of the composition.

The term “about” in relation to a numerical value x is optional andmeans, for example, x+10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encaphalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal derived materials.

Accordingly, the following embodiments are conceivable from the aboveand the examples:

A1. A bulk preparation of a surfactant, characterized in that thesurfactant contains fewer aggregates than the surfactant not subjectedto prefiltration with a filter having a pore size of between 5-50 μm;

-   -   wherein the surfactant is a nonionic surfactant; and,    -   wherein the bulk preparation has a volume of at least 10 liters.        A2. The bulk preparation of embodiment A1, wherein the        surfactant is an anhydrous surfactant.        A3. The bulk preparation of any one of the preceding        embodiments, having a volume of at least 20 liters, 50 liters,        at least 100 liters, at least 125 liters, at least 150 liters,        at least 175 liters, at least 200 liters, at least 250 liters,        or at least 300 liters.        A4. The bulk preparation of any one of the preceding        embodiments, characterized in that the bulk preparation of a        surfactant contains a carbonyl level that is lower than that of        the surfactant not subjected to the pre-filtration.        A5. The bulk preparation of any one of the preceding        embodiments, wherein the nonionic surfactant is selected from        the group consisting of:

TWEEN® 20 (polysorbate 20), TWEEN® 40 (polysorbate 40), TWEEN® 60(polysorbate 60), TWEEN® 80 (polysorbate 80), TRITON™ X-100, IGEPALCA-630, Nonidet P-40, PLURONIC® F-68, PLURONIC® F-88, and PLURONIC®F-127 (poloxamers), and Brij 35 (polyoxyethylene alkyl ether).

A6. A liquid composition comprising an aqueous component and apre-filtered surfactant,

wherein the pre-filtered surfactant is obtained from the bulkpreparation of any one of the preceding embodiments.

A7. The liquid composition of embodiment A6, further comprising an oilcomponent.

A8. The liquid composition of embodiment A6 or A7, wherein the liquidcomposition is an emulsion.

A9. The liquid composition of embodiment A8, wherein the liquidcomposition is an oil-in-water emulsion or a water-in-oil emulsion.

A10. The liquid composition of embodiment A7, wherein the oil componentis selected from the group consisting of:

saturated fatty acids, unsaturated fatty acids, short-chain fatty acids,medium-chain fatty acids, long-chain fatty acids, very long-chain fattyacids, and sterol precursors.

A11. The liquid composition of embodiment of embodiment A10, wherein theoil component is or comprises squalene.

A12. The liquid composition of embodiment A6, wherein the liquidcomposition comprises a surfactant selected from the group consisting ofpolysorbates.

A13. The liquid composition of embodiment A12, wherein the surfactant ispolysorbate 80.

A14. A method for preparing the liquid composition, the methodcomprising the steps of:

pre-filtering a surfactant with a filter having an average pore size of5-50 micron at room temperature so as to form a pre-filtered surfactant;and,

combining the pre-filtered surfactant with an aqueous component so as toform the liquid composition of any one of embodiments A6-A13.

A15. A method for preparing the liquid composition, the methodcomprising the steps of:

providing a prefiltered surfactant according to any one of embodimentsA1-A5; and,

combining the pre-filtered surfactant with an aqueous component so as toform the liquid composition of any one of embodiments A6-A13.

A16. The method of embodiment A14 or A15, wherein the liquid compositionis a pharmaceutical composition comprising at least one API.

A17. The method of embodiment A16, wherein the liquid compositionfurther comprises an oil component.

A18. The method of embodiment A16 or A17, further comprising the stepsof:

emulsifying the liquid composition so as to form a first emulsion;

subjecting the first emulsion to high shear force and high pressure soas to form a second emulsion; and,

filtering the second emulsion, so as to form a uniform emulsion.

A19. The method of embodiment A18, wherein the first emulsion ischaracterized in that a first average oil droplet size is 5000 nm orless, and wherein the number of oil droplets having a size of >1.2 μm inthe first emulsion is 5×10¹¹/ml or less.

A20. The method of embodiment A18 or A19, wherein the second emulsion ischaracterized in that a second average oil droplet size is 500 nm orless, and wherein the number of oil droplets having a size of >1.2 μm inthe second emulsion is 5×10¹⁰/ml or less.

A21. The method of any one of embodiments A18-A20, wherein the uniformemulsion is characterized in that a third average oil droplet size isbetween about 80 and 220 nm, and wherein the number of oil dropletshaving a size of >1.2 μm in the uniform emulsion is 5×10⁸/ml or less.A22. The method of embodiment A21, wherein the uniform emulsioncomprises squalene, polysorbate 80 and sorbitan trioleate.A23. The method of embodiment A22, wherein the uniform emulsioncomprises about 4.3% squalene, about 0.5% polysorbate 80 and about 0.48%sorbitan trioleate by weight.

Specifically, the present invention relates to the followingembodiments:

B1. A process for preparing an oil-in-water emulsion comprising stepsof: (a) filtering polysorbate 80 through a filter having a pore sizebetween 5-50 μm, to provide purified polysorbate 80; and (b) combiningthe purified polysorbate 80 with an oil component to provide theemulsion.B2. The process of embodiment B1, wherein step (a) comprises: filteringnon-aqueous polysorbate 80 through a polypropylene filter having a poresize between 5-50 μm.B3. The process of any preceding embodiment, wherein the purifiedpolysorbate 80 is combined with an aqueous material between steps (a)and (b).B4. The process of any preceding embodiment, wherein step (b) compriseshomogenization to form a homogenized emulsion.B5. The process of any preceding embodiment, wherein step (b) comprisesmicrofluidization to form a microfluidized emulsion.B6. The process of embodiment B4 or embodiment B5, wherein step (b)comprises sterile filtration of the homogenized or microfluidizedemulsion.B7. The process of any preceding embodiment, wherein the oil-in-wateremulsion is a vaccine adjuvant including squalene.B8. The process of embodiment B7, wherein the oil-in-water emulsioncomprises squalene, the polysorbate 80, and sorbitan trioleate, e.g. 5%squalene, 0.5% polysorbate 80 and 0.5% sorbitan trioleate (by volume).B9. The process of any preceding embodiment, wherein the oil-in-wateremulsion has less than 0.85 ppm acetaldehyde.B10. The process of any preceding embodiment, wherein the oil-in-wateremulsion comprises squalene and the emulsion has less than 661 picogramsof polychlorinated biphenyls (PCBs) per gram of squalene (toxicequivalent TEQ).B11. A method for preparing (i) a vaccine composition, comprisingpreparing an oil-in-water emulsion according to any one of embodimentsB1 to B10 and combining the emulsion with an antigen, or (ii) a vaccinekit comprising preparing an oil-in-water emulsion according to any oneof embodiments B1 to B10 and packaging the emulsion into a kit as a kitcomponent together with an antigen component.B12. The method of embodiment B11, wherein the antigen is an influenzavirus antigen; for example, wherein the combination of the emulsion andthe antigen forms a vaccine composition and wherein the vaccinecomposition includes about 15 μg, about 10 μg, about 7.5 μg, about 5 μg,about 3.8 μg, about 1.9 μg, about 1.5 μg of hemagglutinin per influenzavirus strain.

This invention is further illustrated by the following examples.

EXAMPLES

MF59 emulsion adjuvant with squalene, polysorbate 80, sorbitan trioleateand citrate buffer was prepared using the methods disclosed inReferences 10 to 12, with: preparation of a squalene/sorbitan triolatemixture and a solution of polysorbate 80 in citrate buffer; formation ofa first emulsion by homogenization of these components using “type I”then “type II” circulation; formation of a second emulsion bymicrofluidisation (again using “type II” circulation) with an IXC-APMZ-channel arrangement, to reduce oil droplet diameter relative to thefirst emulsion; and filtration of the microfluidised emulsion through ahydrophilic double-layer PES membrane. For the “type II” circulation theemulsion was passed between two containers, but after the finalhomogenization or microfluidisation it was collected into a third(fresh) container. Batch sizes were 50 or 250 liters.

Two different types of non-aqueous polysorbate 80 were used. Withreference to table 1 below, batch numbers 2111-2118 used polysorbate 80which was not filtered prior to being used, whereas batch numbers 2119onwards used polysorbate 80 which was prefiltered through a 20 μmpolypropylene fleece filter capsule (SARTOPURE™ PP 2 MIDICAP™, 20 μm) atroom temperature with a peristaltic pump, before being mixed withcitrate buffer. When using a fresh SARTOPURE™ PP 2 MIDICAP™ 20 μmfilter, the first 200 ml were discarded.

Table 1 shows the effect of this pre-filtration on the amount (ppm) ofacetone, acetaldehyde and formaldehyde in the batches of final MF59emulsion adjuvant which were produced. The thick line in table 1, underrow 2118, indicates the batch at which prefiltration of the anhydrouspolysorbate 80 was commenced. All of the batches below this line weremanufactured using a method in which the anhydrous polysorbate 80 wasprefiltered. Acetaldehyde levels above the line are all above 0.85 ppm,whereas levels below the line are all less than 0.70 ppm.

FIG. 1 shows a graphical depiction of the data in table 1 (includingfurther batches). This FIGURE shows in clear visual terms that batchesprior to batch number 2119 (i.e., those without a prefiltration step)have a higher acetaldehyde content than those produced after batchnumber 2119, (i.e., those for which the prefiltration step wasincluded). Although the results show batch-to-batch variability, theinventor believes that the prefiltration contributes to the reducedcarbonyl levels, and that the process is more robust by usingprefiltered material.

TABLE 1 Average Number of Sorbitan Polysorbate Particle Large ChargeSqualene Trioleate 80 Size Paricles Acetone Acetaldehyde Formaldehyde549 . . . 010 [36; 42] [4.1; 6.3] [4.1; 6.3] [135, 175] 1 × 10⁷ ≤1.5≤2.6 ≤0.2 2111 40.1 4.4 4.4 164 594890 0.22 0.89 0.03 2112 39.7 4.5 4.5175 464840 0.22 0.87 003 2113 39 4.4 4.4 171 444140 0.18 0.86 0.03 211438.5 4.3 4.3 162 358150 0.22 0.86 0.03 2115 39 4.6 4.5 160 537910 0.240.86 0.03 2116 39.1 4.4 4.4 166 314300 0.21 0.85 003 2117 38.7 4.7 4.5161 600830 0.28 0.86 0.03 2118 39.8 4.7 4.5 166 880760 0.12 0.85 0.032119 39.3 4.6 4.4 166 695530 0.06 0.67 0.03 2120 39.5 4.6 4.5 170 4820500.06 0.39 0.04 2121 39.9 4.1 4.2 161 792860 0.06 0.4 0.04 2122 38.9 4.34.2 172 500990 0.08 0.39 0.04 2123 39.4 4.8 4.7 162 509460 0.06 0.4 0.042124 39.3 4.6 4.4 163 518690 0.29 0.45 0.05 2125 38.6 4.8 4.6 1681092500 0.36 0.44 0.05 2126 38.3 4.5 4.4 167 409460 0.22 0.43 0.05 212737.6 4.4 4.4 169 1196670 0.21 0.43 0.05 2128 38.3 4.5 4.4 167 3347800.18 0.44 0.05 2129 38.2 4.6 4.4 168 534930 0.2 0.43 0.06 2130 38.1 4.44.5 168 434100 0.12 0.42 0.05 2131 38.3 4.4 4.4 174 321950 0.11 0.420.05 2132 39.7 4.6 4.5 172 418630 0.11 0.43 0.05 2133 39.6 4.5 4.5 175524520 0.06 0.45 0.05 2134 39.6 4.5 4.5 165 431690 0.06 0.47 0.06 213539.5 4.5 4.6 164 428420 0.07 0.49 0.06

REFERENCES

-   [1] WO90/14837.-   [2] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203.-   [3] Podda (2001) Vaccine 19: 2673-2680.-   [4] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell &    Newman) Plenum Press 1995 (ISBN 0-306-44867-X).-   [5] Vaccine Adjuvants: Preparation Methods and Research Protocols    (Volume 42 of Methods in Molecular Medicine series). ISBN:    1-59259-083-7. Ed. O'Hagan.-   [6] New Generation Vaccines (eds. Levine et al.). 3rd edition, 2004.    ISBN 0-8247-4071-8.-   [7] Kishore et al, (2011) Pharm Res 25:1194-1210.-   [8] Maggio (2012), J. Excipients and Food Chem 3(2): 45-53.-   [9] Yeo et al, (1992) Lipids 27(1):50-53.-   [10] WO2011/067669-   [11] WO2011/067673-   [12] WO2011/067672-   [13] Light Scattering from Polymer Solutions and Nanoparticle    Dispersions (W. Schartl), 2007. ISBN: 978-3-540-71950-2.-   [14] O'Hagan (2007) Expert Rev Vaccines 6(5):699-710.-   [15] EP B 2029170-   [16] Garcon et al. (2012) Expert Rev Vaccines 11:349-66.-   [17] WO2008/043774.-   [18] WO2010/023551-   [19] Brito et al. (2011) Vaccine 29:6262-6268.-   [20] WO94/26683.-   [21] He et al. (2002) J Agric Food Chem 50:368-72.-   [22] WO2011/141819-   [23] U.S. Pat. No. 8,092,813.-   [24] WO2007/052155.-   [25] Hoffmann et al. (2002) Vaccine 20:3165-3170.-   [26] Subbarao et al. (2003) Virology 305:192-200.-   [27] Liu et al. (2003) Virology 314:580-590.-   [28] Ozaki et al. (2004) J. Virol. 78:1851-1857.-   [29] Webby et al. (2004) Lancet 363:1099-1103.-   [30] WO97/37000.-   [31] Brands et al. (1999) Dev Biol Stand 98:93-100.-   [32] Halperin et al. (2002) Vaccine 20:1240-7.-   [33] Tree et al. (2001) Vaccine 19:3444-50.-   [34] Kistner et al. (1998) Vaccine 16:960-8.-   [35] Kistner et al. (1999) Dev Biol Stand 98:101-110.-   [36] Bruhl et al. (2000) Vaccine 19:1149-58.-   [37] Pau et al. (2001) Vaccine 19:2716-21.-   [38] WO01/22992.-   [39] Hehme et al. (2004) Virus Res. 103(1-2):163-71.-   [40] Treanor et al. (1996) J Infect Dis 173:1467-70.-   [41] Keitel et al. (1996) Clin Diagn Lab Immunol 3:507-10.-   [42] Williamson et al. (2006) Infection and Immunity 74: 961-7.-   [43] Loukas et al. (2005) PLoS Med 2(10): e295.-   [44] EPA 0139417.-   [45] Harper et al. (2004) Lancet 364(9447):1757-65.-   [46] J Toxicol Clin Toxicol (2001) 39:85-100.-   [47] Demicheli et al. (1998) Vaccine 16:880-884.-   [48] Stepanov et al. (1996) J Biotechnol 44:155-160.-   [49] Banzhoff (2000) Immunology Letters 71:91-96.-   [50] WO02/097072.-   [51] Greenbaum et al. (2004) Vaccine 22:2566-77.-   [52] Zurbriggen et al. (2003) Expert Rev Vaccines 2:295-304.-   [53] Piascik (2003) J Am Pharm Assoc (Wash D.C.). 43:728-30.-   [54] Mann et al. (2004) Vaccine 22:2425-9.-   [55] Halperin et al. (1979) Am J Public Health 69:1247-50.-   [56] Herbert et al. (1979) J Infect Dis 140:234-8.-   [57] Chen et al. (2003) Vaccine 21:2830-6.

The various features and embodiments of the present invention, referredto in individual sections above apply, as appropriate, to othersections, mutatis mutandis. Consequently, features specified in onesection may be combined with features specified in other sections, asappropriate.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The invention claimed is:
 1. A process for preparing a pharmaceuticallyacceptable composition comprising steps of: (a) filtering a polysorbate80 through a filter membrane having a pore size between 5-50 μm, toprovide purified polysorbate 80 as a filtrate; and (b) using thepurified polysorbate 80 to formulate the pharmaceutically acceptablecomposition.
 2. The process of claim 1, wherein the purified polysorbate80 contains less than 0.85 ppm acetaldehyde.
 3. The process of claim 1,wherein step (a) comprises: filtering non-aqueous polysorbate 80 througha polypropylene filter having a pore size between 5-50 μm.
 4. Theprocess of claim 1, wherein the purified polysorbate 80 is a bulkpreparation of purified polysorbate 80 having a volume of at least 50liters.